Colorectal cancer is the second most common cause of cancer-related mortality in the United States, and 80 percent of patients who die of colorectal cancer have liver metastases. Since current treatments for metastatic colon cancer have achieved only limited success, there is an important need to develop novel and more effective treatment strategies. Our group has used suicide gene therapy based on the herpes simplex virus thymidine kinase gene plus ganciclovir to successfully treat colon cancer liver metastases in a syngeneic animal model. We have also demonstrated that placing the suicide gene under the control of the carcinoembryonic antigen (CEA) promoter affords greater tissue specificity. In addition, we have developed a binary gene expression construct in which the GAL4/VP16 transcription factors are placed under the control of the CEA promoter. This permitted the delivery of a higher dose of suicide gene-containing vector with better tumor cell killing and less toxicity. Although CEA is a useful prototype tumor-associated gene, its expression by many normal cells, particularly biliary epithelium, may result in unacceptable toxicity and limit its clinical utility. The identification of a gene that is more intestine-specific, while maintaining tumor-specificity, should offer the opportunity for greater therapeutic efficacy. Trefoil factor family 3 (TFF3; formerly called intestinal trefoil factor, ITF) is a promising candidate in this regard. TFF3 is primarily expressed by cells of the small and large intestine with no expression in the liver and very restricted expression in other normal organs. Evidence to date indicates that TFF3 expression is highly conserved during the progression from adenoma to carcinoma in the colon. TFF3 has been shown to phosphorylate b-catenin, thereby disrupting cell-cell adhesion and enabling cells to become motile which may explain the role of TFF3 in normal cell migration to heals mucosal wounds, but also for cancer cell invasion and metastasis. We have shown that all human colon cancer liver metastases studied express the TFF3 gene. Therefore, TFF3 appears to be an ideal candidate gene that has both tumor- and intestinal-specificity. The aims of this study are to: 1) Identify elements in the TFF3 promoter that confer colon-specific gene expression; 2) Create and test suicide gene constructs driven by the TFF3 promoter in vitro; and 3) Test the anti-tumor efficacy and toxicity of TFF3-suicide gene constructs in vivo. The use of the TFF3 gene to drive expression of suicide genes offers a novel approach which should result in more targetted, safer gene therapy for metastatic colon cancer.